The Cumulative Effect At The Cellular Level In The Leaves Of Solanum Nigrum L.during The Remediation Of Cd Contamination

At present,China is facing a serious problem of cadmium（Cd）pollution.Phytoremediation based on hyperaccumulators is a hot research topic because of its economic and environmental advantages.However,hyperaccumulators have shortcomings such as slow growth rate and long growth cycle,which limits their application.Genetic modification is a powerful means to enhance plant remediation performance.However,the mechanisms of cadmium absorption,accumulation and detoxification at the molecular level are still not fully clear,which makes the application of genetic modification enhanced phytoremediation slow.Vacuolar compartmentation is the main way for hyperaccumulators to accumulate Cd and avoid disadvantages.Current studies have shown that Cd will enter the vacuole first after entering the cell and be stored in the vacuole.However,it is still uncertain whether the process of Cd entering the vacuole is merely a simple isolation process or a process that simultaneously induces the transfer or depletion of related substances inside and outside the vacuole.How does the chelation mechanism outside the vacuole affect the morphology of Cd involved in vacuolar compartmentation,and does the presence of Cd inside the vacuole alter the distribution of oxidative stress products and antioxidants produced outside the vacuole?These questions are not yet known.The purpose of studying these problems is to establish more abundant vacuolar compartmentation models and provide a more complete theoretical basis for genetic improvement.Based on this,this study explored the physiological and biochemical response to Cd and the accumulation of Cd to determine the enrichment performance of the leaves of S.nigrum.Subsequently,the combined Cd and dissolved Cd²⁺inside and outside the vacuoles were quantified,and the distribution of Cd²⁺was visualized using subcellular separation and fluorescent probe-fluorescence microscopy technology.Meanwhile,the ROS,GSH and other indicators in protoplasts and vacuoles were quantitatively and visually studied to explore the relationship between Cd-GSH-ROS,so as to further understand the role of the vacuole in chelating and antioxidant detoxification.The main results are as follows:（1）Tissue level:The seedlings of S.nigrum were treated with 0,5,25 and 50μM Cd for30 days.The results showed that with the increase of Cd concentration,the growth of S.nigrum decreased continuously,but the decreasing amplitude decreased sharply.Cd stress resulted in the decrease of pigment content in the leaves of S.nigrum and had a significant concentration effect.From the point of concentration and content of accumulation,all tissues of S.nigrum have strong capacity to absorb and store Cd.With the increase of Cd treatment concentration,the transfer coefficient increased obviously,and the accumulation limit of the underground part was reached at 50μM Cd treatment,but the aboveground part was not.The accumulation concentration of Cd in the above-ground part was far more than 100 mg kg^-1DW with each treatment,which further confirmed the enrichment performance of the above-ground part of S.nigrum.（2）Single cell level（vacuolar sequestration and chelation）:The protoplasts and vacuoles in the leaves of S.nigrum were isolated and purified,and Cd²⁺in the single protoplast or vacuole was innovatively quantified by fluorescence probe-fluorescence microscopy combined with flame atomic absorption spectrometry（AAS）.Based on this method,the distribution and proportion of dissolved Cd²⁺and combined Cd were accurately calculated.It is confirmed that vacuoles are the main storage sites for Cd,and Cd mainly exists in the combined form.In addition,it was found a limit to the Cd absorption and accumulation in the protoplasts and vacuoles.With the increase of Cd treatment concentration,the proportion of Cd in vacuoles remained at a certain level（about 83%）,and Cd²⁺began to appear in large quantities outside the vacuoles at 50μM Cd treatment,and the mass concentration of Cd in protoplasts remained basically stable(73.81mg L^-1～77.46 mg L^-1),which demonstrated the critical ability of vacuole sequestration and Cd storge in cell.At the same time,the content and distribution of GSH were measured,and the relationship between GSH and Cd was explored.It was found that GSH content negatively correlated with the combined Cd outside the vacuole.（3）Single-cell level（oxidative stress and antioxidant）:The responses of ROS,GSH and oxidized glutathione（GSSG）in protoplasts and vacuoles under Cd stress were quantitatively studied.It was found that there were ROS transported from outside the vacuole in the vacuole,as well as ROS scavenging by GSH.The evidences are as follows:With the variation of the Cd²⁺and GSH content in vacuoles,the vacuolar ROS level nearly always fluctuates within a certain range.The lack of vacuolar GSH without Cd stress might explain the highest proportion of ROS observed in vacuoles under this condition.The inverse relationship（R²=0.8693）of the proportion of vacuolar GSH and the proportion of vacuolar ROS suggested that the transfer of GSH into vacuoles was related to the relative decrease in the proportion of ROS in vacuoles.In addition,this study confirmed that Cd stress can significantly increase vacuolar GSSG,and vacuoles sequestrate GSSG in large quantities to maintain the balance of GSH/GSSG in the sensitive compartment outside the vacuole.（4）Single-cell level（overall analysis）:This study solved the problems raised.Cd sequestration into vacuoles,as opposed to the single transport of Cd into vacuoles,is an intelligent and active process associated with the chelation and antioxidant-mediated detoxification.The vacuoles separated the most toxic Cd²⁺in large quantities to minimize the damage of Cd stress on plants.The large participation of GSH in chelating detoxification is an important reason for the existence of the main form of combined Cd.Cd-induced ROS can be cleared by GSH and oxidize GSH to GSSG,which then enters the vacuole in large quantities and reduced or decomposed to synthesize GSH.The increased GSH in the vacuole is further involved in the chelating detoxification of Cd²⁺and the antioxidant detoxification of ROS,which forms a cyclic process.In conclusion,this study provided a more complete description of the vacuolar compartmentation process,and provided an important mechanism support and basis for guiding the genetic modification of hyperaccumulators.